Diagnostic composition for the detection of peroxidatively active substances in body fluids

ABSTRACT

Test strips are provided for detecting even small amounts of blood or other peroxidatively active substances in body fluids; the strips comprising a carrier containing a hydroperoxide, at least one chromogen and, as a stabilizer, at least one phosphoric or phosphonic acid amide of the formula WHEREIN R1 is a dimethylamino, alkoxy, aryloxy, alkyl, aryl or Nmorpholine radical; and R2 and R3, which may be the same or different, are dimethylamino or N-morpholine radicals.

United Etates Eatent Rittersdorf et al.

[75] Inventors: Walter Rittersdorf; Hans-Georg Rey; Peter Rieckmann, allof Mannheim-Waldhof, Germany 'Assigneei ehr p s Me uhin Gmbkl,

Mannheim, Germany [22] Filed: July 3, 1973 [21] Appl. No.: 376,249

[30] Foreign Application Priority Data July 18, 1972 Germany 2235127[52] U.S. Cl. 23/230 B, 23/253 TP, 252/400 A,

260/2477 D [51] Int. Cl.....G01n 31/22, GOln 33/16, B01j1/16 [58] Fieldof Search 23/230 B, 253 TP; 252/400 A, 186, 408; 260/247.7 D; 195/1035[56] References Cited UNITED STATES PATENTS 3,252,762 5/1966 Adams, Jr.et al 23/230 B 3,290,117 12/1966 Adams, Jr. et al. 23/230 B Dec. 10,1974 Primary ExaminerMorris O. Wolk Assistant ExaminerArnold TurkAttorney, Agent, or FirmBurgess, Dinklage & Sprung [5 7] ABSTRACT Teststrips are provided for detecting even small amounts of blood or otherperoxidatively active substances in body fluids; the strips comprising acarrier containing a hydroperoxide, at least one chromogen and, as astabilizer, at least one phosphoric or phosphonic acid amide of theformula wherein R is a dimethylamino, alkoxy, aryloxy, alkyl, aryl orN-morpholine radical; and R and R which may be the same or different,are

dimethylamino or N-morpholine radicals.

23 Claims, No Drawings DIAGNOSTIC COMPOSITION FOR THE DETECTION OFPEROXIDATIVELY ACTIVE SUBSTANCES IN BODY FLUIDS The present invention isconcerned with stable test strips for the detection of blood and otherperoxidateactive substances in body fluids.

The detection of small amounts of blood, which are not visible to thenaked eye, in urine, feces or vomit, is very important for the diagnosisof hemorrhages in the stomach, intestines and urinary tract. Suchhemorrhages are caused, for example, by tumors, ulcers or inflammationsof the organs in question. Furthermore, free hemoglobin can also occurin the urine and plasma due to the influence of certain hemolytictoxins. Blood and hemoglobin are peroxidate-active, i.e., they liberateoxygen from hydroperoxides and transfer it to certain acceptors. Otherperoxidate-active substances also occur in leukocytes and bacteria. Thedetection of these substances is important for the diagnosis of diseasesand infections of the kidneys and urinary tract. Myoglobin, which isalso peroxidateactive, is found, for example, in the urine after acardiac infarct. Blood occurs especially frequently in the urine whencalculi are present in the bladder or kidneys.

Their peroxidate-activity is especially suitable for a sensitivedetection of all of these substances. The oxygen liberated from ahydroperoxide can be transferred to a chromogen which is oxidized to acolored material and thus indicates the presence of the peroxidateactivesubstance. This reaction has already been used for quite a long time inmedicinal and forensic analysis, especially for the detection of blood.It is usually carried out in test tubes or by spot tests, hydrogenperoxide usually being used as the hydroperoxide (oxidation agent). Aschromogen, there is preferably used benzidine, o-tolidine or leucomalachite green.

ln view of the great importance which rapid tests have recentlyachieved, tests of this type have also been developed in various waysfor the detection of blood in body fluids. Rapid tests are usuallyabsorbent carriers, preferably papers, which have been impregnated withall of the reagents necessary for the detection reaction and, aftersimply dipping into the body fluid to be tested, show a color reaction.Test papers of this type are described, for example, in U.S. Pat. Nos.3,012,976 and 3,092,464. There are here used, as oxidation agents,organic hydroperoxides, for example cumol hydroperoxide or p-menthanehydroperoxide. These test papers are said to have a sensitive reactionand, especially with regard to blood, possess a greater specificity thanrapid tests with inorganic hydroperoxides.

As is to be expected, these test papers, on which the oxidation agentand chromogen are in very close contact with one another, are notstable. In German Pat. No. 1,265,453, there is described a method ofseparating the two components from one another by encapsulation of theorganic hydroperoxide. For the encapsuling, there can be used gum arabicand gelatine, which have been cross-linked by dialdehydepolysaccharides.

This process is complicated and subject to disturbances. Thus, forexample, expensive machines, such as homogenisers, are needed.Furthermore, long drying times make a continuous impregnation processconsiderably more difficult We have now found that stable test papers,which are easy to produce, are obtained in accordance with theinvention.

Essentially the invention comprises test strips having hydroperoxidesapplied thereto together with at least one phosphoric or phosphonic acidamide of the general formula wherein R, is a dimethylamino, alkoxy,aryloxy, alkyl, aryl or N-morpholine radical and R and R which may bethe same or different, are dimethylamino or N- morpholine radicals. I

The alkoxy or alkyl radicals R, preferably contain up to 10 carbonatoms. The aryl or aryloxy radicals can be, for example, phenyl ornaphthyl radicals which are optionally substituted by halogen atoms orlower alkyl or alkoxy radicals.

The test papers according to the present invention can be produced bydissolving a hydroperoxide in a solvent, preferably in a water-alcoholmixture, together with an-amide of general formula (I) and optionallytogether with a buffer and a wetting agent and impregnating filter paperwith this solution followed by drying. When using readily volatilesolvents, drying only takes a few minutes. Thus, according to thisprocess, filter papers can be continuously impregnated and dried withoutdifficulty.

For the detection of peroxidate-active substances in feces, it is alsopossible to incorporate the reagents with the stabilizers used accordingto the present invention, into a water-stable film in the mannerdescribed in German Pat. No. 1,598,153. This has the advantage that thesurface of the test strip can, for reading off the color reaction, becleaned simply by wiping it.

Thus, the present invention provides a test strip for the detection ofperoxidate-active substances in body fluids, comprising a carriercontaining a hydroperoxide, at least one chromogen and a compound ofgeneral formula (I), as stabilizer.

Most of the compounds of general formula (I) are known and can beprepared by known and simple processes. Some of the compounds of generalformula (I) are liquids at ambient temperature and some of them are verysparingly soluble in water. It is obvious that solid and readilywater-soluble compounds of general formula (I) are preferred for thestabilization of test strips, since these provide especially usefulcommercial products. When the compounds of general formula (I), forexample in the case of hexamethyl phosphoric acid triamide, are liquid,then the test strip has an oily and somewhat unsatisfactory appearance,whereas waterinsoluble compounds of general formula (I) can lead to anundesirable hydrophobing effect.

The stability of the test strips according to the present invention ismost surprising since substances, the action of which, as stabilizersfor hydrogen peroxide, is known, for example, urea, mannitol,acetanilide and the like, here show no stabilizing action whatsoever.

The compounds of general formula (I) are preferably used in a ratio ofat least one mole per mole hydroperoxide group, a two to fourfold excesshaving proved to be especially useful.

Compounds of general formula (I) in which one or more of the radicals RR and R contain an aromatic ring, somewhat reduce the reactivity of thehydroperoxides so that they are only chosen as stabilizers forespecially active hydroperoxides.

As hydroperoxides, there can be used the common representatives whenthey are not too volatile, for example, tert.-butyl hydroperoxide. Thesolid compounds 2,5-dimethylhexane-2,S-dihydroperoxide, tetralinehydroperoxide and diisopropyl-benzene dihydroperoxide have proved to beespecially useful but liquid compounds, such asdiisopropyl-benzene-hydroperoxide, cumol hydroperoxide, p-menthanehydroperoxide and pinane hydroperoxide can also be used.

The hydroperoxides can be used in amounts of 0.5- g., preferably of l-3g., per 100 ml. of impregnation solution Further components of the testpapers according to the present invention include chromogens, buffers,wetting agents, thickening agents and possibly activators.

As chromogens, there can be used all those which can easily be oxidizedto give deep-colored compounds. These include, in particular, benzidineand its homologues, especially o-tolidine. Furthermore, the heterocyclicazines according to German Pat. No. l,648,840 have also proved to beuseful.

The chromogens can be used in amounts of from 0.05-5 g., preferably of0.2-1 .0 g., per 100 ml. impregnation solution.

As buffers, there can be used, for example, citrate, phosphate,phthalate or succinate buffers, the pH value and capacity thereof beingselected in such a manner that, after dipping the test strip into thebody fluid, there is obtained a pH value of 4-7 and preferably of 5-6.

It is also advantageous to add to the formulation small amounts, forexample about 0.05-0.5 g. per 100 ml., of a complex former, for examplesodium metaphosphate or ethylene-diamine tetraacetic acid, falselypositive reaction which can be caused by traces of metals thereby beingavoided.

Since the test papers can tend to bleed due to the relatively largeamounts of water-soluble substances present therein, it can be desirableto add to the formulation thickening agents, for example methylcellulose and especially of gelatine, in amounts of about 0.5-5 g. per100 ml.

In order to increase the sensitivity of the reaction, socalledactivators can also be added. These include, for example, quinoline andderivatives thereof according to German Pat. No. 1,242,905.

As wetting agents, there are preferably used longchain organic sulfatesor sulfonates, for example sodium dodecylbenzene sulfonate, dioctylsodium sulfosuccinate or sodium lauryl sulfate, which, as is known,stabilize radical cations, such as oxidized o-tolidine.

For the production of the test strips according to the presentinvention, there can be used absorbent carriers, for example filterpaper, cellulose or synthetic fiber fleeces, which are impregnated withsolutions of the reagents in readily volatile solvents. The impregnationis preferably carried out in two steps. First, impregnation is carriedout with a solution which contains an amide of general formula (I), aswell as a hydroperoxide, a wetting agent, a buffer and possibly athickening agent. Thereafter, impregnation is carried out with asolution of the indicator and possibly of the activator..When usinghydrophobic monohydroperoxides, it is advisable to apply the buffer andthe hydroperoxide to the carrier in separate impregnation steps.

For the production of water-stable films, all the reagents, togetherwith a stabilizer of general formula (I), are introduced into a solutionor dispersion of a filmforming substance, for example, a polyvinyl esteror polyamide and mixed homogeneously. The mixture is then applied in athin layer to a substrate, for example a synthetic resin carrier, anddried.

' The test strips according to the present invention are, after drying,cut up into strips and preferably sealed between a synthetic resin filmand a fine-mesh material in the manner described in German Pat. No. 2,118,455.

The following Examples are given for the purpose of illustrating thepresent invention:

EXAMPLE 1 A solution is prepared containing the following components:

dioctyl sodium sulphosuccinate 200 g. ethylenediaminc-tetraacetic acid,l0 g. disodium salt 2,5-dimethyl-hexane-2,5dihydro- 160 g. peroxidephosphoric acid trimorpholide 1270 g. citrate buffer (1.2 molar, pH5.25) 3.5 liters ethanol 3.0 liters distilled water ad 10.0 liters Thesolution is placed into a trough which is provided with deflectionrollers. Thereafter, a filter paper strip is drawn continuously at aspeed of about 2 meters/minute through the solution and dried in adrying canal with a length of 15 meters in a current of air with atemperature of 40C.

The paper pre-treated in this manner is further impregnated in the samemanner with a 0.3% solution of o-tolidine in toluene which contains 0.2%quinine as activator.

There is obtained a pure white test paper which does not becomediscolored when stored in the usual manner.

When the phosphoric acid trimorpholide is omitted from theabove-described formulation then, even during the second impregnation, apale green coloration occurs which, after a few days, intensifies to astrong green-brown coloration.

Practically the same properties are shown by test papers which, insteadof phosphoric acid trimorpholide, contain equimolar amounts of thefollowing amides: phosphoric acid dimethylamide dimorpholide, phosphoricacid ethyl ester dimorpholide and ethane phosphonic acid dimorpholide.

EXAMPLE 2 Filter paper is impregnated with the following solution I anddried for about l5 minutes at C. in a drying cabinet:

Solution l diisopropyl-benzene hydroperoxide (50%) 3.5 g. benzenephosphonic acid dimorpholide 6.! g. sodium dodecyl-benzene sulfonate 2 0g. methanol ad 100.0 ml.

Thereafter, the filter paper was impregnated with the followingsolutions and dried at 40C.:

The test papers thus obtained were characterized by an outstandingstability and do not become discolored.

Practically the same properties are shown by test papers which, insteadof benzene phosphonic acid dimorpholide, contain equimolar amounts ofthe following amides: benzene phosphonic acid bis-(dimethylamide) orphosphoric acid phenyl ester dimorpholide.

It will be understood that the specification and examples areillustrative but not limitative of the present invention and that otherembodiments within the spirit and scope of the invention will suggestthemselves to those skilled in the art.

What is claimed is:

1. Test strip for the detection of peroxidatively active substances inbody fluids, comprising a carrier containing a hydroperoxide, at leastone chromogen and, as a stabilizer, at least one phosphoric orphosphonic acid amide of the formula wherein I R, is dimethylamino,alkoxy, aryloxy, alkyl, aryl or N-morpholino and contains not more thancarbon atoms; R and R are individually selected from dimethylamino andN-morpholino. 2. Test strip as claimed in claim 1, wherein R, in theformula is dimethylamino.

3. Test strip as claimed in claim 1, wherein R in the formula is loweralkoxy.

4. Test strip as claimed in claim 1, wherein R in the formula is aryloxyof up to 10 carbon atoms.

5. Test strip as claimed in claim 1, wherein R, in the formula is loweralkyl.

6. Test strip as claimed in claim 1, wherein R in the formula is aryl ofup to 10 carbon atoms.

7. Test strip as claimed in claim 1, wherein R, in the formula isN-morpholino.

8. Test strip as claimed in claim 1, wherein R in the formula isdimethylamino.

9. Test strip as claimed in claim 1, wherein R in the formula isN-morpholino.

10. Test strip as claimed in claim 1, wherein R in the formula isdimethylamino.

11. Test strip as claimed in claim 1, wherein R in the formula isN-morpholino.

12. Test strip as claimed in claim 1, wherein said acid amide isphosphoric acid trimorpholide.

13. Test strip as claimed in claim 1, wherein said acid amide isphosphoric acid dimethylamide dimorpholide.

14. Test strip as claimed in claim 1, wherein said acid amide isphosphoric acid ethyl ester dimorpholide.

15. Test strip as claimed in claim 1, wherein said acid amide is ethanephosphonic acid dimorpholide.

16. Test strip as claimed in claim 1, wherein the carrier is anabsorbent material impregnated with the reagents.

17. Test strip as claimed in claim 1, wherein the carrier is awater-stable film containing the reagents.

18. Test strip as claimed in claim 1, wherein there is at least one moleof said acid amide per mole of hydroperoxide.

19. Test strip as claimed in claim 1, wherein a buffer is additionallycontained in the test strip.

20. Test strip as claimed in claim 19, wherein sufficient buffer isprovided to give a pH of4 to 7 to the test strip in use.

21. Test strip as claimed in claim 1, also containing at least one of acomplex former, a thickening agent, an activator and a wetting agent.

22. Method of detecting a peroxidatively active substance in a bodyfluid, which method comprises contacting a test sample with a test stripas claimed in claim 1.

23. Method as claimed in claim 22, wherein said peroxidatively activesubstance is blood.

1. TEST STRIP FOR THE DETECTION OF PEROXIDATIVELY ACTIVE SUBSTANCES INBODY FLUIDS, COMPRISING A CARRIER CONTAINING A HYDROPEROXIDE, AT LEASTONE CHROMOGEN AND, AS A STABILIZER, AT LEAST ONE PHOSPHORIC ORPHOSPHONIC ACID AMIDE OF THE FORMULA
 2. Test strip as claimed in claim1, wherein R1 in the formula is dimethylamino.
 3. Test strip as claimedin claim 1, wherein R1 in the formula is lower alkoxy.
 4. Test strip asclaimed in claim 1, wherein R1 in the formula is aryloxy of up to 10carbon atoms.
 5. Test strip as claimed in claim 1, wherein R1 in theformula is lower alkyl.
 6. Test strip as claimed in claim 1, wherein R1in the formula is aryl of up to 10 carboN atoms.
 7. Test strip asclaimed in claim 1, wherein R1 in the formula is N-morpholino.
 8. Teststrip as claimed in claim 1, wherein R2 in the formula is dimethylamino.9. Test strip as claimed in claim 1, wherein R2 in the formula isN-morpholino.
 10. Test strip as claimed in claim 1, wherein R3 in theformula is dimethylamino.
 11. Test strip as claimed in claim 1, whereinR3 in the formula is N-morpholino.
 12. Test strip as claimed in claim 1,wherein said acid amide is phosphoric acid trimorpholide.
 13. Test stripas claimed in claim 1, wherein said acid amide is phosphoric aciddimethylamide dimorpholide.
 14. Test strip as claimed in claim 1,wherein said acid amide is phosphoric acid ethyl ester dimorpholide. 15.Test strip as claimed in claim 1, wherein said acid amide is ethanephosphonic acid dimorpholide.
 16. Test strip as claimed in claim 1,wherein the carrier is an absorbent material impregnated with thereagents.
 17. Test strip as claimed in claim 1, wherein the carrier is awater-stable film containing the reagents.
 18. Test strip as claimed inclaim 1, wherein there is at least one mole of said acid amide per moleof hydroperoxide.
 19. Test strip as claimed in claim 1, wherein a bufferis additionally contained in the test strip.
 20. Test strip as claimedin claim 19, wherein sufficient buffer is provided to give a pH of 4 to7 to the test strip in use.
 21. Test strip as claimed in claim 1, alsocontaining at least one of a complex former, a thickening agent, anactivator and a wetting agent.
 22. Method of detecting a peroxidativelyactive substance in a body fluid, which method comprises contacting atest sample with a test strip as claimed in claim
 1. 23. Method asclaimed in claim 22, wherein said peroxidatively active substance isblood.